The DF3/MUC1 antigen is a high molecular weight glycoprotein which is overexpressed in human breast cancers. In addition, aberrant glycosylation of the DF3 protein core has led to identification of certain epitopes, such as DF3-P, which are selectively expressed by transformed mammary epithelium. Studies have demonstrated that these normally cryptic epitopes are recognized in selected patients by cytotoxic T cells and by antibodies. Taken together, these findings have indicated that breast cancer-associated antigens are expressed in humans, but the T cell response to these epitopes is insufficient in most individuals to prevent the progression of disease. Activation of T cells requires the delivery of two signals by antigen presenting cells (APCs). The first signal is mediated by interaction of the T cell receptor with specific antigenic peptide presented on the APC in the context of the major histocompatibility complex (MHC). The second signal, termed costimulatory, is also delivered by the APC through members of the B7 family. The absence of costimulating molecules and thereby functional activation of mature T cells may result in induction of tolerance to tumor antigens. The concept of vaccine therapy and immune surveillance would ideally be tested in patients who have only micrometastases. A substantial portion of patients with metastatic breast cancer who are treated with high dose chemotherapy and autologous bone marrow transplantation (STAMP) are rendered grossly disease free, yet their risk of subsequent progression is high. In this project, we propose to develop a vaccine therapy strategy against the tumor-associated DF3/MUC1 antigen. Initial preclinical studies will be followed by a phase I study designed to determine the optimal vaccine dose in patients with metastatic breast cancer. These studies will serve as the basis for a phase I-II trial in metastatic breast cancer patients in complete remission after STAMP in which we will define toxicity and immune response to vaccination. These pilot trials will provide data from which prospective, randomized studies can be designed to test whether vaccination against DF3/MUC1 substantially improves the long-term outcomes of patients with breast cancer who have minimal residual disease. The Specific Aims are: 1) To induce active specific immunotherapy in preclinical models using recombinant vaccinia viruses containing either DF3/MUC1, B7-1, B7-2 or combinations thereof. 2) To perform a Phase I/II study in patients with metastatic breast cancer to determine the optimal dose of vaccinia virus that expresses DF3/MUC1 with or without B7-1 or B7-2 as determined from preclinical studies. 3) To perform a Phase I/II study in patients with metastatic breast cancer who are in complete remission after STAMP to determine the optimal schedule for vaccination with the DF3/MUC1 virus with or without B7 molecules as determined in Specific Aims 1 and 2.